Nerve growth factor composition and powder injection

ABSTRACT

The present invention discloses a nerve growth factor composition and an injection powder comprising a nerve growth factor, a stabilizer, a pH buffer, and water, wherein the stabilizer is at least one of sorbitol, dextran, trehalose, raffinose and mannitol. By using a carbohydrate or sugar alcohol as a stabilizer, the present invention can avoid the potential risk resulting from the presence of viruses or other unknown components in albumin. In addition to the nerve growth factor, only the stabilizer and the pH buffer are contained, without conventional surfactant and supporting agent, increasing safety and significantly reducing incidence of the adverse reactions. The present invention not only provides a protective effect on mNGF, but also can ensure good stability of hNGF and rhNGF in the preparation, transportation and storage processes, and has better medication safety and quality control.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a U.S. National Phase of International PatentApplication Serial No. PCT/CN2016/079570, entitled “NERVE GROWTH FACTORCOMPOSITION AND POWDER INJECTION,” filed on Apr. 18, 2016. InternationalPatent Application Serial No. PCT/CN2016/079570 claims priority toChinese Patent Application No. 201510188969.0, filed on Apr. 21, 2015.The entire contents of each of the above-cited applications are herebyincorporated by reference for all purposes.

TECHNICAL FIELD

The present invention relates to a nerve growth factor composition andan injection powder, and belongs to the field of pharmaceutical biology.

BACKGROUND ART

Nerve growth factor (NGF) is a nerve cell growth regulator with thedouble biological functions of neuronal nutrition and neurite growthpromotion, and it has an important regulatory effect on the development,differentiation, growth, regeneration and functional property expressionof the central and peripheral neurons. NGF contains three subunits, α,β, γ, wherein the β subunit is an active region, formed by two singlechains composed of 118 amino acids by a non-covalent bond. In 1953, theItalian scientist Levi-Montalcini discovered NGF and won the NobelPrize. At present, there are a number of NGF products which appear onthe market all over the world, and they are clinically used mainly forthe treatment of neurological dysplasia, including amblyopia, neuroma,various nerve injury and neurological diseases and other diseases.

NGF is similar to other protein drugs; since the protein has a shorthalf life, the spatial conformation of the protein easily changes andthus results in protein denaturation when exposed to extreme temperatureand humidity conditions, or by influenced by physical and chemicalfactors; the denatured protein will lose its original biologicalactivity; in addition, because the protein often tends to adhere to asolid surface, in the filling process, part of the protein will adhereto the inner wall of the container, resulting in the loss of activeingredients. In order to ensure its biological activity, there is a needto add a stabilizer to prevent the loss of the biological activity.

Generally, albumin is widely used in various biological products as anexcellent stabilizer and as a cake forming agent. However, since albuminis mainly derived from human blood, placental blood, and the blood maycarry some of the unknown components which are not easy to be detected,the NGF composition as a non-sterile preparation which is sterilized,may easily be contaminated; at the same time, the long-term and wideapplications of albumin are also susceptible to blood supply constraintsand production costs; again, in the determination of the contents of theintermediates and preparation of finished product, albumin may interferewith a relatively small amount of NGF and thus affect the productquality management. Therefore, in order to avoid the above problems, itis necessary to find a stable albumin-free NGF composition.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a nerve growthfactor composition and an injection powder which not only protectmouse-derived nerve growth factor (mNGF) but also can ensure the goodstability of human-derived nerve growth factor (hNGF) and recombinanthuman nerve growth factor (rhNGF) in the preparation, transportation andstorage processes, and have better clinical medication safety andquality controllability.

The present invention provides a nerve growth factor compositionconsisting of a nerve growth factor, a stabilizer, a pH buffer and awater;

-   -   said stabilizer is at least one of sorbitol, dextran, trehalose,        raffinose and mannitol.

In the above nerve growth factor composition:

-   -   a mass-volume concentration of said nerve growth factor is 10        μg/mL-100 μg/mL;    -   a mass-volume concentration of said stabilizer is 30 mg/mL-120        mg/mL;    -   the pH buffer maintains said nerve growth factor composition at        a pH value of 6.0 to 7.4;    -   solvent is water.

The addition of a stabilizer in the above nerve growth factorcomposition may avoid or reduce the aggregation and depolymerization ofproteins caused in the preparation or storage process, and the term“stabilizer” refers to a substance which prevents the active ingredientfrom aggregating or depolymerizing in an aqueous solution, and inaddition to the function of stability, the stabilizer can also be usedas a supporting agent to improve the product formability, with otherfunctions being not excluded;

-   -   in the above nerve growth factor composition, preferably, the        mass-volume concentration of said stabilizer is 70 mg/mL-120        mg/mL, and more preferably, the mass-volume concentration of        said stabilizer is 70 mg/mL;    -   in the above nerve growth factor composition, said stabilizer        may specifically be any one of the following 1) to 8): 1)        sorbitol and mannitol, 2) sorbitol, dextran and mannitol, 3)        trehalose and mannose, 4) raffinose and mannitol, 5)        sorbitol, 6) trehalose, 7) sorbitol, raffinose and mannitol, 8)        dextran and mannitol.

In the above nerve growth factor composition, said nerve growth factoris a nerve cell growth regulator with the double biological functions ofneuronal nutrition and neurite growth promotion, and it has an importantregulatory effect on the development, differentiation, growth,regeneration and functional property expression of the central andperipheral neurons;

-   -   in the above nerve growth factor composition, preferably, the        mass-volume concentration of said nerve growth factor is 40        μg/mL-60 μg/mL, preferably, the mass-volume concentration of        said nerve growth factor is 50 μg/mL-60 μg/mL, more preferably,        the mass-volume concentration of said nerve growth factor is 60        μg/mL;    -   in the above nerve growth factor composition, said nerve growth        factor is a mouse-derived nerve growth factor (mNGF), a        human-derived nerve growth factor (hNGF), or a recombinant human        nerve growth factor (rhNGF).

In the above nerve growth factor composition, in order to ensure thatthe composition has the greatest biological activity, it is generallynecessary to control an optimum pH range. The optimum pH range for thisstability needs to be determined at the time of formulation screening,usually using an influencing factor test (illumination, hightemperature, high humidity), accelerated and long-term stability testsand other methods. After the determination of the formulation, thecomposition in the production and storage process must be maintained atits optimum pH range. Since the buffer has good buffering capacity, therelative stability of the product pH can be maintained in a certainrange; therefore, a buffer is often added for the control of pH value inthe formulation;

-   -   in the above nerve growth factor composition, the molar        concentration of said pH buffer is 10 mM-50 mM, preferably, the        molar concentration of said pH buffer is 20 mM-30 mM, and more        preferably, the molar concentration of said pH buffer is 25 mM;    -   in the above nerve growth factor composition, preferably, the pH        value of said nerve growth factor composition is 6.81-6.87, more        preferably, the pH value of said nerve growth factor composition        is 6.81;    -   said pH buffer is at least one of a phosphate, a citrate, an        acetate, a histidine hydrochloride and an arginine        hydrochloride, and may specifically be any one of the following        1)-6): 1) a phosphate, 2) a citrate, 3) an acetate, 4) a        histidine hydrochloride, 5) a arginine hydrochloride, and 6) a        citrate and arginine hydrochloride, wherein the molar ratio of        the citrate to arginine hydrochloride in 6) is 3:2.

In the above nerve growth factor composition, said water is water forinjection.

In the above nerve growth factor composition, the concentration of eachcomponent may be any one of the following 1) to 15):

-   -   1) the nerve growth factor: 10-50 μg/mL, the stabilizer: 30-64.5        mg/mL, the pH buffer: 10 mM-25 mM;    -   2) the nerve growth factor: 60-100 μg/mL, the stabilizer: 55-70        mg/mL, the pH buffer: 25 mM-50 mM;    -   3) the nerve growth factor: 40-80 μg/mL, the stabilizer: 40-65        mg/mL, the pH buffer: 20 mM-30 mM;    -   4) the nerve growth factor: 40-80 μg/mL, the stabilizer: 60-80        mg/mL, the pH buffer: 20 mM-30 mM;    -   5) the nerve growth factor: 40-80 μg/mL, the stabilizer:        64.5-120 mg/mL, the pH buffer: 20 mM-30 mM;    -   6) the nerve growth factor: 40-60 μg/mL, the stabilizer: 70-120        mg/mL, the pH buffer: 20 mM-30 mM;    -   7) the nerve growth factor: 10 μg/mL, the stabilizer: 30 mg/mL,        the pH buffer: 10 mM;    -   8) the nerve growth factor: 40 μg/mL, the stabilizer: 40 mg/mL,        the pH buffer: 20 mM;    -   9) the nerve growth factor: 50 μg/mL, the stabilizer: 55 mg/mL,        the pH buffer: 25 mM;    -   10) the nerve growth factor: 60 μg/mL, the stabilizer: 80 mg/mL,        the pH buffer: 30 mM;    -   11) the nerve growth factor: 80 μg/mL, the stabilizer: 64.5        mg/mL, the pH buffer: 50 mM;    -   12) the nerve growth factor: 100 μg/mL, the stabilizer: 70        mg/mL, the pH buffer: 50 mM;    -   13) the nerve growth factor: 100 μg/mL, the stabilizer: 80        mg/mL, the pH buffer: 50 mM;    -   14) the nerve growth factor: 100 μg/mL, the stabilizer: 120        mg/mL, the pH buffer: 50 mM;    -   15) the nerve growth factor: 60 μg/mL, the stabilizer: 70 mg/mL,        the pH buffer: 25 mM.

In the above nerve growth factor composition, said nerve growth factorcomposition is specifically maintained at a pH value of 6.80-7.00, 6.80,6.81, 6.83, 6.84, 6.85, 6.87, 6.90, 6.92 or 7.00.

The present invention further provides a method for preparing a nervegrowth factor composition injection powder, comprising the step of:lyophilizing the above nerve growth factor composition to obtain thenerve growth factor composition injection powder.

The present invention also provides a nerve growth factor compositioninjection powder for injection administration.

In the above nerve growth factor composition injection powder, the watercontent of said injection powder may be 1.0%-3.0%, preferably 1.0%-2.8%,specifically 1.1%, 1.2%, 1.3%, 1.6%, 1.7%, 2.6% or 2.8%; the pH value ofsaid injection powder may be 6.82-7.08, preferably 6.87-7.08,specifically 6.82, 6.85, 6.87, 6.96, 7.02, 7.03 or 7.05.

The use of the above nerve growth factor compositions in the treatmentof nerve injury or the preparation of a medicament for the treatment ofnerve injury is also within the scope of the present invention. Saidnerve growth factor composition may specifically be a nerve growthfactor composition injection powder. The nerve injury may be an opticnerve injury, and the cause of the injury may be a first injury, a caraccident, a physical hit injury or an eye explosive injury.

The present invention further provides a method of treating nerve injurycomprising the step of: administering an effective amount of a nervegrowth factor composition to a patient with the nerve injury. Said nervegrowth factor composition may specifically be a nerve growth factorcomposition injection powder. The administration may specifically beintramuscular injection. The nerve injury may be an optic nerve injury,and the cause of the injury may be a first injury, a car accident, aphysical hit injury or an eye explosive injury.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the change curve of the average content of NGF over time inthe NGF compositions and injection powders prepared in the Examples andthe reference preparation, under the accelerated condition (25° C., RH60±10%), wherein −1 represents the injection powders, and −2 representsthe compositions.

FIG. 2 shows the change curve of the average activity over time of theNGF compositions and injection powders prepared in the Examples and thereference preparation, under the accelerated condition (25° C., RH60±10%), wherein −1 represents the injection powders, and −2 representsthe compositions.

FIG. 3 shows the change curve of the average content of NGF over time inthe NGF compositions and injection powders prepared in the Examples andthe reference preparation, under the long-term stability condition (6±2°C.), wherein −1 represents the injection powders, and −2 represents thecompositions.

FIG. 4 shows the change curve of the average activity over time of theNGF compositions and injection powders prepared in the Examples and thereference preparation, under the long-term stability condition (6±2°C.), wherein −1 represents the injection powders, and −2 represents thecompositions.

DETAILED DESCRIPTION OF EMBODIMENTS

The experimental methods used in the following Examples are conventionalmethods unless otherwise specified.

The materials, reagents and the like used in the following Examples arecommercially available, unless otherwise specified.

The mNGF and hNGF stock solutions used in the following Examples aresupplied by the Staidson (Beijing) Biopharmaceuticals Co., Ltd., therhNGF stock solution is supplied by the Sino Biological Inc., and therest of the excipients are all injection grade unless otherwisespecified.

In order to find a substitute for albumin as a stabilizer for NGF, inthe present invention, a large number of exploratory experiments havebeen carried out, in which sorbitol, dextran, trehalose, raffinose andmannitol are used as a stabilizer of the present product, to prepare thevarious lyophilized NGF composition sterile injection powders, and themoisture, pH, osmotic pressure, content and activity of the finishedproduct after lyophilization are determined.

Example 1. Preparation of NGF Composition and Injection Powder

The NGF composition and the injection powder are prepared as follows:

-   -   a formulation amount of a phosphate is weighed according to the        formulation composition in Table 1, an appropriate amount of        water for injection is added and stirred to complete        dissolution, then a formulation amount of trehalose is added and        stirred to complete dissolution, a hNGF stock solution is added,        the pH is adjusted to pH 6.84 as shown in Table 1 with NaOH or        phosphoric acid, water for injection is added to the volume of        the scale, after having been mixed uniformly, the mixture is        filtered through a 0.22 μm microfiltration membrane into a        sterile container to prepare a composition having a hNGF        concentration of about 60 μg/mL.

The above hNGF composition is subpackaged into a borosilicate glassinjection bottle at 0.63±0.03 mL/bottle to prepare an NGF compositionsample. The sample is lyophilized, to prepare a sterile powder for hNGFinjection, i.e., a hNGF injection powder.

Example 2. Preparation of NGF Composition and Injection Powder

The NGF composition and the injection powder are prepared as follows:

-   -   a formulation amount of an arginine hydrochloride is weighed        according to the formulation composition in Table 1, an        appropriate amount of water for injection is added and stirred        to complete dissolution, then formulation amounts of sorbitol,        dextran and mannitol are added and stirred to complete        dissolution, a hNGF stock solution is added, the pH is adjusted        to pH 6.80 as shown in Table 1 with arginine or HCl, water for        injection is added to the volume of the scale, after having been        mixed uniformly, the mixture is filtered through a 0.22 μm        microfiltration membrane into a sterile container to prepare a        composition having a hNGF concentration of about 40 μg/mL.

The above hNGF composition is subpackaged into a borosilicate glassinjection bottle at 0.63±0.03 mL/bottle to prepare an NGF compositionsample. The sample is lyophilized, to prepare a sterile powder for hNGFinjection, i.e., a hNGF injection powder.

Example 3. Preparation of NGF Composition and Injection Powder

The NGF composition and the injection powder are prepared as follows:

-   -   a formulation amount of an acetate is weighed according to the        formulation composition in Table 1, an appropriate amount of        water for injection is added and stirred to complete        dissolution, then formulation amounts of trehalose and mannitol        are added and stirred to complete dissolution, a rhNGF stock        solution is added, the pH is adjusted to pH 6.87 as shown in        Table 1 with NaOH or acetic acid, water for injection is added        to the volume of the scale, after having been mixed uniformly,        the mixture is filtered through a 0.22 μm microfiltration        membrane into a sterile container to prepare a composition        having a rhNGF concentration of about 50 μg/mL.

The above rhNGF composition is subpackaged into a borosilicate glassinjection bottle at 0.63±0.03 mL/bottle to prepare an NGF compositionsample. The sample is lyophilized, to prepare a sterile powder for rhNGFinjection, i.e., a rhNGF injection powder.

Example 4. Preparation of NGF Composition and Injection Powder

The NGF composition and the injection powder are prepared as follows:

-   -   a formulation amount of a histidine hydrochloride is weighed        according to the formulation composition in Table 1, an        appropriate amount of water for injection is added and stirred        to complete dissolution, then formulation amounts of raffinose        and mannitol are added and stirred to complete dissolution, a        mNGF stock solution is added, the pH is adjusted to pH 6.85 as        shown in Table 1 with histidine or HCl, water for injection is        added to the volume of the scale, after having been mixed        uniformly, the mixture is filtered through a 0.22 μm        microfiltration membrane into a sterile container to prepare a        composition having a mNGF concentration of about 10 μg/mL.

The above mNGF composition is subpackaged into a borosilicate glassinjection bottle at 0.63±0.03 mL/bottle to prepare an NGF compositionsample. The sample is lyophilized, to prepare a sterile powder for mNGFinjection, i.e., a mNGF injection powder.

Example 5. Preparation of NGF Composition and Injection Powder

The NGF composition and the injection powder are prepared as follows:

-   -   formulation amounts of a citrate and an arginine hydrochloride        are weighed according to the formulation composition in Table 1,        an appropriate amount of water for injection is added and        stirred to complete dissolution, then a formulation amount of        sorbitol is added and stirred to complete dissolution, a hNGF        stock solution is added, the pH is adjusted to pH 6.92 as shown        in Table 1 with NaOH or citric acid, water for injection is        added to the volume of the scale, after having been mixed        uniformly, the mixture is filtered through a 0.22 μm        microfiltration membrane into a sterile container to prepare a        composition having a hNGF concentration of about 50 μg/mL.

The above hNGF composition is subpackaged into a borosilicate glassinjection bottle at 0.63±0.03 mL/bottle to prepare an NGF compositionsample. The sample is lyophilized, to prepare a sterile powder for hNGFinjection.

Example 6. Preparation of NGF Composition and Injection Powder

The NGF composition and the injection powder are prepared as follows:

-   -   a formulation amount of a citrate is weighed according to the        formulation composition in Table 1, an appropriate amount of        water for injection is added and stirred to complete        dissolution, then formulation amounts of sorbitol, trehalose and        mannitol are added and stirred to complete dissolution, a hNGF        stock solution is added, the pH is adjusted to pH 6.81 as shown        in Table 1 with NaOH or citric acid, water for injection is        added to the volume of the scale, after having been mixed        uniformly, the mixture is filtered through a 0.22 μm        microfiltration membrane into a sterile container to prepare a        composition having a hNGF concentration of about 60 μg/mL.

The above hNGF composition is subpackaged into a borosilicate glassinjection bottle at 0.63±0.03 mL/bottle to prepare an NGF compositionsample. The sample is lyophilized, to prepare a sterile powder for hNGFinjection, i.e., a hNGF injection powder.

Example 7. Preparation of NGF Composition and Injection Powder

The NGF composition and the injection powder are prepared as follows:

-   -   a formulation amount of an acetate is weighed according to the        formulation composition in Table 1, an appropriate amount of        water for injection is added and stirred to complete        dissolution, then formulation amounts of sorbitol, raffinose and        mannitol are added and stirred to complete dissolution, a mNGF        stock solution is added, the pH is adjusted to pH 7.00 as shown        in Table 1 with NaOH or acetic acid, water for injection is        added to the volume of the scale, after having been mixed        uniformly, the mixture is filtered through a 0.22 μm        microfiltration membrane into a sterile container to prepare a        composition having a mNGF concentration of about 50 μg/mL.

The above mNGF composition is subpackaged into a borosilicate glassinjection bottle at 0.63±0.03 mL/bottle to prepare an NGF compositionsample. The sample is lyophilized, to prepare a sterile powder for mNGFinjection, i.e., a mNGF injection powder.

Example 8. Preparation of NGF Composition and Injection Powder

The NGF composition and the injection powder are prepared as follows:

-   -   a formulation amount of a citrate is weighed according to the        formulation composition in Table 1, an appropriate amount of        water for injection is added and stirred to complete        dissolution, then formulation amounts of sorbitol, and mannitol        are added and stirred to complete dissolution, a rhNGF stock        solution is added, the pH is adjusted to pH 6.90 as shown in        Table 1 with NaOH or citric acid, water for injection is added        to the volume of the scale, after having been mixed uniformly,        the mixture is filtered through a 0.22 μm microfiltration        membrane into a sterile container to prepare a composition        having a rhNGF concentration of about 100 μg/mL.

The above rhNGF composition is subpackaged into a borosilicate glassinjection bottle at 0.63±0.03 mL/bottle to prepare an NGF compositionsample. The sample is lyophilized, to prepare a sterile powder for rhNGFinjection, i.e., a rhNGF injection powder.

Example 9. Preparation of NGF Composition and Injection Powder

The NGF composition and the injection powder are prepared as follows:

-   -   a formulation amount of a histidine hydrochloride is weighed        according to the formulation composition in Table 1, an        appropriate amount of water for injection is added and stirred        to complete dissolution, then formulation amounts of dextran and        mannitol are added and stirred to complete dissolution, a hNGF        stock solution is added, the pH is adjusted to pH 6.81 as shown        in Table 1 with histidine or HCl, water for injection is added        to the volume of the scale, after having been mixed uniformly,        the mixture is filtered through a 0.22 μm microfiltration        membrane into a sterile container to prepare a composition        having a hNGF concentration of about 80 μg/mL.

The above hNGF composition is subpackaged into a borosilicate glassinjection bottle at 0.63±0.03 mL/bottle to prepare an NGF compositionsample. The sample is lyophilized, to prepare a sterile powder for hNGFinjection, i.e., a hNGF injection powder.

Example 10. Preparation of NGF Composition and Injection Powder

The NGF composition and the injection powder are prepared as follows:

-   -   a formulation amount of a phosphate is weighed according to the        formulation composition in Table 1, an appropriate amount of        water for injection is added and stirred to complete        dissolution, then formulation amounts of trehalose and mannitol        are added and stirred to complete dissolution, a hNGF stock        solution is added, the pH is adjusted to pH 6.83 as shown in        Table 1 with NaOH or phosphoric acid, water for injection is        added to the volume of the scale, after having been mixed        uniformly, the mixture is filtered through a 0.22 μm        microfiltration membrane into a sterile container to prepare a        composition having a hNGF concentration of about 50 μg/mL.

The above hNGF composition is subpackaged into a borosilicate glassinjection bottle at 0.63±0.03 mL/bottle to prepare an NGF compositionsample. The sample is lyophilized, to prepare a sterile powder for hNGFinjection, i.e., a hNGF injection powder.

TABLE 1 The amounts of components in Examples 1-10 Formulation Amountsof components in the Examples composition Use Example 1 Example 2Example 3 Example 4 Example 5 Example 6 Example 7 Example 8 Example 9Example 10 mNGF stock Active 10 50 solution ingredient hNGF stock(μg/mL) 60 40 50 60 80 50 solution rhNGF stock 50 100 solution SorbitolStabilizer 10 30 10 10 40 trehalose (mg/mL) 70 50 50 60 dextran 4.5 5Raffinose 10 5 mannitol 50 70 50 20 40 25 35 20 phosphate Buffered 25 20acetate salt (mM) 25 25 citrate 30 30 50 histidine 10 20 hydrochloridearginine 30 20 hydrochloride pH in 6.84 6.80 6.87 6.85 6.92 6.81 7.006.90 6.81 6.83 Examples

Example 11. Performance Test of the NGF Compositions Prepared inExamples 3, 6 and 7 and the Injection Powders of Examples 1-10

(1) Test of Appearance, Moisture, Osmotic Pressure, pH, Content andActivity

The NGF compositions prepared in Examples 3, 6 and 7, the NGFcomposition injection powders prepared in Examples 1-10 and a referencepreparation (mouse nerve growth factor for injection, trade name:Sutaisheng, manufactured by the Staidson (Beijing) BiopharmaceuticalsCo., Ltd.) are observed for the appearances, respectively, and theirmoistures, osmotic pressures, pHs, contents and activities aredetermined. The method of determining the NGF content is carried outaccording to the method described in Example 1 in Patent Application No.200510130348.3, entitled “Method for Determining the Content of NerveGrowth Factor”. The activity test method is determined by a cell method,and the detailed method of operation is carried out according to themethod of Example 1 in Patent Publication No. CN 103376248A, entitled“Method for Quantitative Determination of Nerve Growth Factor Activity”.The experimental results are shown in Table 2.

TABLE 2 Test results of test items in the Examples Test results of thetest items Osmotic Average Average Moisture pressure content activitySample Appearance (%) (mOsm/kg) pH (%) (U/bottle) Reference white loose0.9 102 7.02 99.08 19313 preparation solid blocks Example 1 white loose2.6 143 7.08 99.39 26009 solid blocks Example 2 white loose 1.6 151 6.82100.13 17727 solid blocks Example 3 white loose 2.5 183 7.05 98.77 19939solid blocks Example 3 Colorless N/A 183 7.05 98.51 19901 clear liquidExample 4 white loose 1.2 150 6.87 98.99 8338 solid blocks Example 5white loose 1.1 121 7.03 99.61 17072 solid blocks Example 6 white loose2.8 158 6.87 100.08 24558 solid blocks Example 6 Colorless N/A 158 6.8798.85 24510 clear liquid Example 7 white loose 1.7 155 6.92 98.81 19326solid blocks Example 7 Colorless N/A 155 6.92 98.62 19296 clear liquidExample 8 white loose 1.3 151 6.85 99.24 39175 solid blocks Example 9white loose 1.2 127 6.87 99.52 32137 solid blocks Example 10 white loose2.6 159 6.96 98.38 19723 solid blocks

As can be seen from Table 2, the indexes of the NGF compositions andinjection powders prepared in the present invention all meet the qualitystandard requirements.

(2) Stable pH Range Screening

According to the formulation composition of Example 3, 3 NGFcompositions are formulated, wherein the total amount of acetate is keptconstant and the relative ratio of acetic acid to sodium acetate isadjusted so that the pH values of the intermediate solution NGFcompositions are measured at 6.0, 6.8 and 7.4, respectively, then filledinto penicillin bottles, respectively, and lyophilized to preparesterile injection powder. Samples are taken, and an appropriate amountof sterile water for injection is added to formulate into solutions.Under room temperature condition, the solutions are observed for theappearance and determined for the pH value, the content and the activityat 0, 4, 8, 12 and 24 hours, respectively. The results are shown in thefollowing table.

TABLE 3 Stable pH range screening results of NGF compositions AverageAverage Sample Placing time content activity name (h) Appearance pH (%)(%) Sample 1 0 Colorless clear 6.06 98.78 19122 (pH 6.0) solution 4Colorless clear 6.03 98.45 19658 solution 8 Colorless clear 6.03 98.8818968 solution 12 Colorless clear 6.05 98.25 19152 solution 24 Colorlessclear 6.02 97.61 18829 solution Sample 2 0 Colorless clear 6.80 99.9219328 (pH 6.8) solution 4 Colorless clear 6.82 100.25 19591 solution 8Colorless clear 6.82 99.75 18778 solution 12 Colorless clear 6.84 99.3818592 solution 24 Colorless clear 6.80 99.09 18957 solution Sample 3 0Colorless clear 7.42 99.35 18868 (pH 7.4) solution 4 Colorless clear7.39 99.16 18513 solution 8 Colorless clear 7.41 99.21 18051 solution 12Colorless clear 7.39 98.95 17827 solution 24 Colorless clear 7.42 97.3318381 solution

It can be seen from the above table data that after the three NGFcompositions with different pH values are placed at room temperature for24 h, the indexes all have no significant changes than at 0 h, all meetthe quality standard requirements, indicating that under roomtemperature condition, the sample solutions can remain stable within 24h in a range of pH 6.0-7.4, more preferably at pH 6.8.

(3) Stability Test

A. Accelerated Stability Test

According to the requirements of “Technical Guidelines for Research onStability of Biological Products”, the samples of the referencepreparation (Sutaisheng), the NGF compositions prepared in Examples 3, 6and 7, and the NGF composition injection powders prepared in theExamples are examined for an accelerated stability for 0-6 months underconditions of 25° C., RH 60±10%. The samples are observed for theappearances, and determined for the moistures, osmotic pressures, pHvalues, contents and activities. The results are shown in Table 4 andFIG. 1 (the change curve of average content over time), and FIG. 2 (thechange curve of average activity over time).

TABLE 4 Accelerated stability test results of the NGF compositions, theinjection powders and the reference preparation Osmotic Average AverageTime Moisture pressure content activity (month) Sample Appearance (%)(mOsm/kg) pH (%) (U/bottle) 0 Reference white loose 0.9 102 7.02 99.0819313 preparation solid blocks Example 1 white loose 2.6 143 7.08 99.3926009 solid blocks Example 2 white loose 1.6 151 6.82 100.13 17727 solidblocks Example 3 white loose 2.5 183 7.05 98.77 19939 solid blocksExample 3 Colorless N/A 183 7.05 98.51 19901 clear liquid Example 4white loose 1.2 150 6.87 98.99 8338 solid blocks Example 5 white loose1.1 121 7.03 99.61 17072 solid blocks Example 6 white loose 2.8 158 6.87100.08 24558 solid blocks Example 6 Colorless N/A 158 6.87 98.85 24510clear liquid Example 7 white loose 1.7 155 6.92 98.81 19326 solid blocksExample 7 Colorless N/A 155 6.92 98.62 19296 clear liquid Example 8white loose 1.3 151 6.85 99.24 39175 solid blocks Example 9 white loose1.2 127 6.87 99.52 32137 solid blocks Example 10 white loose 2.6 1596.96 98.38 19723 solid blocks 1 Reference white loose 1.0 105 6.87 97.3819157 preparation solid blocks Example 1 white loose 2.7 146 7.07 99.0226127 solid blocks Example 2 white loose 1.4 155 6.85 99.62 17352 solidblocks Example 3 white loose 2.5 183 7.03 98.35 19989 solid blocksExample 3 Colorless N/A 182 7.02 98.05 19786 clear liquid Example 4white loose 1.3 152 6.87 98.50 8703 solid blocks Example 5 white loose1.2 126 7.02 98.23 17257 solid blocks Example 6 white loose 1.9 161 6.8399.71 24277 solid blocks Example 6 Colorless N/A 160 6.84 99.39 24102clear liquid Example 7 white loose 1.7 160 6.93 98.25 19509 solid blocksExample 7 Colorless N/A 162 6.93 97.99 19016 clear liquid Example 8white loose 1.4 153 6.86 98.89 38851 solid blocks Example 9 white loose1.2 130 6.86 98.91 31999 solid blocks Example 10 white loose 2.5 1576.85 98.58 19581 solid blocks 2 Reference white loose 1.1 105 7.03 95.6518875 preparation solid blocks Example 1 white loose 2.6 141 7.07 98.7825658 solid blocks Example 2 white loose 1.7 150 6.85 99.22 17188 solidblocks Example 3 white loose 2.4 178 7.07 97.88 19621 solid blocksExample 3 Colorless N/A 177 7.05 97.43 19433 clear liquid Example 4white loose 1.3 152 6.89 97.77 8429 solid blocks Example 5 white loose1.2 123 7.05 97.46 16868 solid blocks Example 6 white loose 2.9 158 6.8999.51 24099 solid blocks Example 6 Colorless N/A 158 6.91 98.95 23858clear liquid Example 7 white loose 1.8 153 6.95 97.64 18989 solid blocksExample 7 Colorless N/A 156 6.95 97.03 18751 clear liquid Example 8white loose 1.2 153 6.85 98.11 38156 solid blocks Example 9 white loose1.3 125 6.88 97.67 31058 solid blocks Example 10 white loose 2.7 1576.97 98.15 19226 solid blocks 3 Reference white loose 1.0 103 7.05 94.1617556 preparation solid blocks Example 1 white loose 2.5 145 7.05 98.4125089 solid blocks Example 2 white loose 1.7 153 6.85 98.79 16859 solidblocks Example 3 white loose 2.6 185 7.03 97.31 19228 solid blocksExample 3 Colorless N/A 183 7.01 96.62 19005 clear liquid Example 4white loose 1.3 152 6.88 96.15 8083 solid blocks Example 5 white loose1.2 124 7.05 95.60 16452 solid blocks Example 6 white loose 2.7 156 6.8999.31 23891 solid blocks Example 6 Colorless N/A 155 6.90 98.66 23641clear liquid Example 7 white loose 1.8 156 6.96 96.88 18442 solid blocksExample 7 Colorless N/A 156 6.95 96.13 18112 clear liquid Example 8white loose 1.4 153 6.88 97.28 37705 solid blocks Example 9 white loose1.3 124 6.89 96.43 30689 solid blocks Example 10 white loose 2.7 1576.99 97.72 18989 solid blocks 6 Reference white loose 1.1 102 7.04 89.3817302 preparation solid blocks Example 1 white loose 2.5 141 7.08 97.7124829 solid blocks Example 2 white loose 1.5 152 6.84 96.26 16695 solidblocks Example 3 white loose 2.6 183 7.06 96.58 18853 solid blocksExample 3 Colorless N/A 181 7.05 95.98 18636 clear liquid Example 4white loose 1.3 151 6.88 94.61 7759 solid blocks Example 5 white loose1.0 122 7.05 93.22 15699 solid blocks Example 6 white loose 2.8 157 6.8899.19 23665 solid blocks Example 6 Colorless N/A 157 6.89 98.39 23453clear liquid Example 7 white loose 1.8 156 6.93 95.23 18058 solid blocksExample 7 Colorless N/A 155 6.92 94.36 17828 clear liquid Example 8white loose 1.4 152 6.88 95.82 36824 solid blocks Example 9 white loose1.2 129 6.89 94.98 30221 solid blocks Example 10 white loose 2.5 1616.98 97.21 18758 solid blocks

As can be seen from FIG. 1, the average content of NGF in the referencepreparation, the NGF compositions prepared in Examples 3, 6 and 7, andthe NGF composition injection powders prepared in the Examples presentsa reduction tendency over time under accelerated conditions (25° C., RH60±10%), wherein the reduction speed in the average content of NGF inthe reference preparation is significantly greater than that of thecompositions of the Examples of the present invention. As can be seenfrom the results in Table 4, the appearances, moistures, pH values andosmotic pressures of the samples are not significantly changed under theaccelerated conditions (25° C., RH 60±10%) for 6 months, but the contentof the reference preparation reduces by about 9.8%, the reduction ratesin the contents of the NGF composition injection powders of Examples 1to 10 are 1.7%, 3.9%, 2.2%, 4.4%, 6.4%, 0.9%, 3.6%, 3.4%, 4.6%, 1.2%,respectively, and the reduction rates in the contents of the NGFcompositions of Examples 3, 6 and 7 are 2.6%, 1.5% and 4.3%,respectively, indicating that the stability of the compositions of theExamples of the present invention is superior to that of the referencepreparation under the accelerated conditions, particularly the stabilityof the compositions of Example 1, Example 3, Example 6 and Example 10 issignificantly superior to the reference preparation, and the stabilityof the composition injection powders is superior to that of thecompositions.

As can be seen from FIG. 2, the average activity of NGF in the referencepreparation, the NGF compositions prepared in Examples 3, 6 and 7, andthe NGF compositions and the NGF composition injection powders preparedin the Examples presents a reduction tendency over time underaccelerated conditions (25° C., RH 60±10%), wherein the activity of thereference preparation reduces rapidly after two months of acceleration.As can be seen from the results in Table 4, the appearances, moistures,pH values and osmotic pressures of the samples are not significantlychanged under accelerated conditions (25° C., RH 60±10%) for 6 months,but the activity of the reference preparation reduces by 10.4%, thereduction rates in the activities of the NGF composition injectionpowders of the Examples 1 to 10 are 4.5%, 5.8%, 5.4%, 6.9%, 8.0%, 3.6%,6.6%, 6.0%, 6.0% and 4.9%, respectively, and the reduction rates in theactivities of the NGF compositions of the Examples 3, 6 and 7 are 6.4%,4.3% and 7.6%, respectively, indicating that the stability of theExamples of the present invention is superior to that of the referencepreparation under the accelerated conditions, particularly the stabilityof the compositions of Example 1, Example 3, Example 6 and Example 10 issignificantly superior to that of the reference preparation, and thestability of the composition injection powders is superior to that ofthe compositions.

B. Long-Term Stability Test

According to the requirements of “Technical Guidelines for Research onStability of Biological Products”, the samples of the referencepreparation (Sutaisheng), the NGF compositions prepared in Examples 3, 6and 7, and the NGF composition injection powders prepared in theExamples are examined for a long-term stability for 0-12 months undercondition of 6±2° C. The samples are observed for the appearances, anddetermined for the moistures, osmotic pressures, pH values, contents andactivities. The results are shown in Table 5, and FIG. 3 (the changecurve of average content over time) and FIG. 4 (the change curve ofaverage activity over time).

TABLE 5 Long-term stability test results of the NGF compositions, theinjection powders and the reference preparation Osmotic Average Averagetime Moisture pressure content activity (month) Sample Appearance (%)(mOsm/kg) pH (%) (U/bottle) 0 Reference white loose 0.9 102 7.02 99.0819313 preparation solid blocks Example 1 white loose 2.6 143 7.08 99.3926009 solid blocks Example 2 white loose 1.6 151 6.82 100.13 17727 solidblocks Example 3 white loose 2.5 183 7.05 98.77 19939 solid blocksExample 3 Colorless N/A 183 7.05 98.51 19901 clear liquid Example 4white loose 1.2 150 6.87 98.99 8338 solid blocks Example 5 white loose1.1 121 7.03 99.61 17072 solid blocks Example 6 white loose 2.8 158 6.87100.08 24558 solid blocks Example 6 Colorless N/A 158 6.87 98.85 24510clear liquid Example 7 white loose 1.7 155 6.92 98.81 19326 solid blocksExample 7 Colorless N/A 155 6.92 98.62 19296 clear liquid Example 8white loose 1.3 151 6.85 99.24 39175 solid blocks Example 9 white loose1.2 127 6.87 99.52 32137 solid blocks Example 10 white loose 2.6 1596.96 98.38 19723 solid blocks 3 Reference white loose 1.0 107 7.03 98.1219578 preparation solid blocks Example 1 white loose 2.5 145 7.05 99.0826128 solid blocks Example 2 white loose 1.6 149 6.83 99.57 17516 solidblocks Example 3 white loose 2.6 181 7.06 98.59 20110 solid blocksExample 3 Colorless N/A 180 7.05 98.16 19768 clear liquid Example 4white loose 1.3 152 6.89 98.26 8507 solid blocks Example 5 white loose1.2 123 7.05 99.05 16938 solid blocks Example 6 white loose 2.7 159 6.89100.35 24388 solid blocks Example 6 Colorless N/A 157 6.91 99.72 24135clear liquid Example 7 white loose 1.8 156 6.95 98.05 19103 solid blocksExample 7 Colorless N/A 155 6.94 97.86 19011 clear liquid Example 8white loose 1.2 153 6.87 98.62 38588 solid blocks Example 9 white loose1.3 129 6.84 98.87 31922 solid blocks Example 10 white loose 2.5 1616.95 98.59 19927 solid blocks 6 Reference white loose 0.9 104 7.04 97.7918853 preparation solid blocks Example 1 white loose 2.5 144 7.05 98.5725883 solid blocks Example 2 white loose 1.7 150 6.84 99.32 17262 solidblocks Example 3 white loose 2.4 182 7.05 98.01 19753 solid blocksExample 3 Colorless N/A 180 7.03 97.55 19433 clear liquid Example 4white loose 1.1 151 6.87 97.59 8158 solid blocks Example 5 white loose1.1 122 7.02 97.53 17025 solid blocks Example 6 white loose 2.9 159 6.8899.68 24121 solid blocks Example 6 Colorless N/A 158 6.89 99.40 23998clear liquid Example 7 white loose 1.8 154 6.96 97.69 18575 solid blocksExample 7 Colorless N/A 153 6.95 97.21 18738 clear liquid Example 8white loose 1.4 152 6.86 98.01 38156 solid blocks Example 9 white loose1.2 128 6.88 97.59 31087 solid blocks Example 10 white loose 2.5 1576.97 98.32 19562 solid blocks 9 Reference white loose 1.0 107 7.03 95.7818620 preparation solid blocks Example 1 white loose 2.6 145 7.07 98.4225321 solid blocks Example 2 white loose 1.6 150 6.84 98.83 17011 solidblocks Example 3 white loose 2.4 181 7.06 97.52 19528 solid blocksExample 3 Colorless N/A 182 7.03 97.03 19102 clear liquid Example 4white loose 1.3 152 6.88 96.4 8025 solid blocks Example 5 white loose1.2 124 7.05 96.15 16581 solid blocks Example 6 white loose 2.8 156 6.8799.43 23997 solid blocks Example 6 Colorless N/A 155 6.88 99.11 23758clear liquid Example 7 white loose 1.6 158 6.92 96.78 18602 solid blocksExample 7 Colorless N/A 157 6.93 96.19 18402 clear liquid Example 8white loose 1.4 153 6.86 97.25 37618 solid blocks Example 9 white loose1.3 129 6.87 96.77 30444 solid blocks Example 10 white loose 2.7 1576.97 97.98 19119 solid blocks 12 Reference white loose 0.9 105 7.0393.55 18192 preparation solid blocks Example 1 white loose 2.5 144 7.0798.06 25076 solid blocks Example 2 white loose 1.6 153 6.82 97.36 16878solid blocks Example 3 white loose 2.5 184 7.05 96.95 19182 solid blocksExample 3 Colorless N/A 184 7.04 96.56 18979 clear liquid Example 4white loose 1.3 152 6.88 95.22 7915 solid blocks Example 5 white loose1.1 122 7.04 94.82 16220 solid blocks Example 6 white loose 2.7 157 6.8899.41 23756 solid blocks Example 6 Colorless N/A 158 6.89 98.97 23553clear liquid Example 7 white loose 1.8 155 6.92 95.90 18403 solid blocksExample 7 Colorless N/A 155 6.93 95.38 18102 clear liquid Example 8white loose 1.4 153 6.86 96.33 37280 solid blocks Example 9 white loose1.2 129 6.88 95.31 30625 solid blocks Example 10 white loose 2.5 1586.96 97.52 18998 solid blocks

As can be seen from FIG. 3, the average content of NGF in the referencepreparation, the NGF compositions prepared in Examples 3, 6 and 7, andthe NGF composition injection powders prepared in the Examples presentsa reduction tendency over time under the long-term stability condition(6±2° C.), wherein the reduction speed in the average content of NGF inthe reference preparation is significantly greater than that of Examplesof the present invention. As can be seen from the results in Table 5,the appearances, moistures, pH values and osmotic pressures of thesamples are not significantly changed under the long-term condition for12 months, but the content of the reference preparation reduces by about5.6%, the reduction rates in the contents of the NGF compositioninjection powders of Examples 1 to 10 are 1.3%, 2.8%, 1.8%, 3.8%, 4.8%,0.7%, 2.9%, 2.9%, 4.2% and 0.9%, respectively, and the reduction ratesin the contents of the NGF compositions of Examples 3, 6 and 7 are 2.0%0.9%, and 3.3%, respectively, indicating that the stability of thecompositions of the Examples of the present invention is superior tothat of the reference preparation under the long-term condition,particularly the stability of the compositions in Example 1, Example 3,Example 6 and Example 10 is significantly superior to the referencepreparation, and the stability of the injection powders is superior tothat of the compositions.

As can be seen from FIG. 4, the average activity of NGF in the referencepreparation, the NGF compositions prepared in Examples 3, 6 and 7, andthe NGF compositions and the NGF composition injection powders preparedin the Examples presents a reduction tendency over time under thelong-term stability condition (6±2° C.), wherein the reduction speed inthe activity of the reference preparation is greater than that ofExamples of the present invention. As can be seen from the results inTable 5, the appearances, moistures, pH values and osmotic pressures ofthe samples are not significantly changed under the long-term conditionfor 12 months, but the activity of the reference preparation reduces by5.8%, the reduction rates in the activities of the NGF compositioninjection powders of the Examples 1 to 10 are 3.6%, 4.8%, 3.8%, 5.1%,5.0%, 3.3%, 4.8%, 4.8%, 4.7% and 3.7%, respectively, and the reductionrates in the activities of the NGF compositions of the Examples 3, 6 and7 are 4.6%, 3.9%, and 6.2%, respectively, indicating that the stabilityof the Examples of the present invention is superior to that of thereference preparation under the long-term condition, particularly thestability of Example 1, Example 3, Example 6 and Example 10 issignificantly superior to that of the reference preparation, and thestability of the injection powders is superior to that of thecompositions.

(4) Clinical Evaluation

A total of 271 patients with optic nerve injury are subjected to a12-weeks clinical trial by the multicenter non-randomized controlledclinical study design; all the subjects are 14 years or older, and areable to communicate well with the investigators, understand and complywith clinical trial requirements, and sign an informed consent.

Subject condition: 14 years or older.

Gender: male or female.

Subject source: 409 patients with optic nerve injury caused by variouscauses from various ophthalmic research units being enrolled.

Injury cause: 136 cases of first injury (136 eyes), 152 cases of caraccident (152 eyes), 73 cases of physical hit injury (73 eyes), 49 casesof eye explosive injury (49 eyes). All cases are not accompanied byeyeball rupture and damage caused by the optic nerve compression due toorbital fractures.

Treatment method: the treatment group is injected with the referencepreparation and the NGF compositions of the present invention byintramuscular injection once a day with each 30 μg, and all cases areadministered continuously for 12 weeks. The placebo group is treatedwith a negative control (with no active ingredient, and the remainingexcipients being the same to those in the NGF compositions).

The treatment group comprises 271 cases (271 eyes), with age of 18 to 63years, and the average age of 34.2 years; 189 males (189 eyes), 82females (82 eyes); 147 eyes in the right eye, 124 eyes in the left eye.The placebo group comprises 138 patients (138 eyes), with age of 13-55years, and the average age of 33.9 years, 96 males (96 eyes), 42 females(42 eyes), 45 eyes in the right eye and 93 eyes in the left eye.

Grouping design: grouping is shown in the following table:

TABLE 6 Grouping of clinical trial and number of subjects Trial groupingand number of subjects (person) Placebo Reference NGF Test group grouppreparation group composition group Fist injury 46 45 45 Car accident 5051 51 Physical hit injury 25 24 24 Explosive injury 17 16 16 Number ofsubjects in 138 135 136 each group Note: for the NGF composition groupin the above table, the nerve growth factor composition prepared inExample 6 is used, the same below.

Clinical common adverse reaction evaluation: the placebo group, thereference preparation group and the NGF composition group areadministered, respectively, and observed for the symptom and incidenceof the adverse reactions in each group.

TABLE 7 NGF clinical common adverse reactions Adverse Reference NGFreaction Placebo group preparation group composition group symptom (N =138) (N = 135) (N = 136) Local pain 3 (2.2) 11 (8.1)*  4 (2.9)*#Urticaria 1 (0.7) 6 (4.4)* 2 (1.5)*# Dizziness 10 (7.2)  7 (5.2)* 3(2.2)*# Insomnia 7 (5.1) 7 (5.2)* 2 (1.5)*# Conjunctival 0 (0)   1(0.7)* 1 (0.7)*  congestion Note: *indicates that there is a significantdifference compared with the placebo group (P < 0.05), #indicates thatthere is a significant difference compared with the referencepreparation group (P < 0.05).

It can be seen from the above table that, compared with the placebogroup, the main adverse reactions of the compositions prepared in thepresent invention are local pain and dizziness, and compared with thereference preparation group, the NGF compositions prepared in thepresent invention can significantly alleviate the adverse reactions,such as local pain, urticaria, dizziness, etc.

Clinical medicine efficacy: the placebo group, the reference preparationgroup and the NGF composition group are administered, respectively, andobserved for the clinical efficacy in each group. The results are shownin the following table.

TABLE 8 Comparison of NGF clinical efficacy Reference Clinical Placebogroup preparation group NGF composition group efficacy (N = 138) (N =135) (N = 136) Recovery 3 (2.2) 85 (63.0) 92 (67.6) Effective 11 (8.0) 21 (15.6) 33 (24.3) Alleviated 9 (6.5) 21 (15.6) 9 (6.6) Ineffective 115(83.3)  8 (5.8) 2 (1.5)

The clinical efficacy in the above table is evaluated using thefollowing clinical efficacy evaluation criteria:

-   -   {circle around (1)} Recovery: the vision recovers to 1.0 or        more, and dark spots in the central visual field disappear;    -   {circle around (2)} Effective: the vision increases by 4 lines        or more, dark spots in the central visual field reduce or the        absolute dark spots become relative dark spots;    -   {circle around (3)} Alleviated: the vision increases by 2 lines        or more, and there is no change in visual field;    -   {circle around (4)} Ineffective: the vision and visual field are        the same as before the treatment, or decline.

It can be seen from the above table that, compared with the placebogroup, the NGF compositions and the injection powders of the presentinvention can be effective in treating optic nerve injury caused byvarious causes, have the comprehensive efficiency of the treatment ofoptic nerve injury that is superior to that of the reference preparationgroup, can significantly reduce the incidence of the adverse reactions,and have good clinical therapeutic effect.

While the present invention has been described in detail by way ofgeneral description, particular embodiments and examples, variousmodifications and improvements may be made by those skilled in the arton the basis of the present invention without departing from the spiritof the invention and such modifications or improvements are all withinthe scope according to the present invention.

INDUSTRIAL APPLICATION

The nerve growth factor compositions and the injection powders preparedin the present invention retain good stability in preparation,transportation and storage processes: (1) in the preparation process:after the nerve growth factor composition injection powders prepared inthe present invention are stored at room temperature for 24 hours, thecontent and activity of the NGF therein have no significant change; (2)compared with the reference preparation, in the conventional (6±2° C.)transportation and storage processes, the NGF composition injectionpowders of the present invention has an NGF content reduction of only0.7%-4.8% and an activity reduction of only 3.3%-5.0%, after beingstored for 12 months, thereby having the excellent stability.

The nerve growth factor compositions and the injection powders preparedin the present invention can significantly reduce the incidence of theadverse reactions in the clinical trial, have good clinical therapeuticeffect, and have better clinical medication safety and qualitycontrollability as compared with the existing reference preparation.

The present invention has the following beneficial effects:

-   -   (1) the nerve growth factor compositions and the injection        powders of the present invention can avoid the potential risk        resulting from the virus or other unknown components carried in        albumin by using a carbohydrate or a sugar alcohol instead of        albumin as a stabilizer.    -   (2) in the nerve growth factor compositions and the injection        powders of the present invention, in addition to the nerve        growth factor as active ingredient, only two components of a        stabilizer and a pH buffer are contained, without conventional        surfactant, supporting agent and the like; so such compositions        have a simple composition, better safety, and a significantly        reduced incidence of the adverse reactions; and the compositions        have definite ingredients, are easily qualitative and        quantitative, and the stabilizer used therein has a high purity,        a wide source and easy long-term mass production, which        facilitate the cost control and the product quality improvement.    -   (3) the nerve growth factor compositions and the injection        powders of the present invention not only have protective effect        on mouse nerve growth factor (mNGF), but also can ensure the        good stability of the human nerve growth factor (hNGF) and the        recombinant human nerve growth factor (abbreviated as rhNGF) in        the preparation, transportation and storage processes, and have        better clinical medication safety and quality controllability.

The invention claimed is:
 1. A nerve growth factor composition, whereinthe nerve growth factor composition consists of nerve growth factor(NGF), a stabilizer, a pH buffer, and a solvent; wherein the stabilizeris sorbitol, trehalose, and mannitol at a mass-volume concentrationratio of 1:5:2, respectively; wherein the solvent is water; wherein thepH buffer is at least one of a citrate, a histidine hydrochloride, andan arginine hydrochloride; and wherein the nerve growth factorcomposition does not contain a surfactant.
 2. The nerve growth factorcomposition of claim 1, wherein: a mass-volume concentration of thenerve growth factor is between 10 μg/mL to 100 μg/mL; a mass-volumeconcentration of the stabilizer is 30 mg/mL to 120 mg/mL; and the pHbuffer maintains said nerve growth factor composition at a pH value of6.0 to 7.4.
 3. The nerve growth factor composition of claim 1, wherein amass-volume concentration of the stabilizer is between 70 mg/mL to 120mg/mL.
 4. The nerve growth factor composition of claim 2, wherein themass-volume concentration of the nerve growth factor is between 40 μg/mLto 60 μg/mL.
 5. The nerve growth factor composition according to claim1, wherein the nerve growth factor is a mouse-derived nerve growthfactor, a human-derived nerve growth factor, or a recombinant humannerve growth factor.
 6. The nerve growth factor composition according toclaim 1, wherein a mole concentration of the pH buffer is between 10 mMto 50 mM.
 7. The nerve growth factor composition of claim 2, wherein:the mass-volume concentration of the nerve growth factor is 60 μg/mL;the mass-volume concentration of the stabilizer is 80 mg/mL; and a moleconcentration of the pH buffer is 30 mM.
 8. A method for preparation ofa nerve growth factor composition injection powder, comprising:lyophilizing the nerve growth factor composition according to claim 1 toobtain the nerve growth factor composition injection powder.
 9. Thenerve growth factor composition injection powder prepared by the methodof claim
 8. 10. The nerve growth factor composition according to claim1, wherein the pH buffer is citrate and arginine hydrochloride.
 11. Thenerve growth factor composition of claim 10, wherein a molar ratio ofcitrate to arginine hydrochloride is 3:2.
 12. The nerve growth factorcomposition according to claim 1, wherein the pH buffer is citrate. 13.The nerve growth factor composition according to claim 1, wherein a moleconcentration of the pH buffer is 30 mM.
 14. A nerve growth factorcomposition, wherein the nerve growth factor composition consists of anerve growth factor, a stabilizer, a pH buffer, and a solvent; whereinthe stabilizer is at least one of sorbitol, dextran, trehalose,raffinose, and mannitol; wherein the solvent is water; wherein the pHbuffer is citrate and arginine hydrochloride at a molar ration of 3:2;and wherein the nerve growth factor composition does not contain asurfactant.
 15. The nerve growth factor composition of claim 14, whereinthe stabilizer is sorbitol, trehalose, and mannitol.
 16. The nervegrowth factor composition of claim 15, wherein the stabilizer is of amass-volume concentration ratio of 1:5:2 of sorbitol, trehalose, andmannitol, respectively.
 17. The nerve growth factor composition of claim14, wherein the stabilizer is sorbitol, dextran, and mannitol.
 18. Thenerve growth factor composition of claim 17, wherein the stabilizer isof a mass-volume concentration ratio of 1:0.45:5 of sorbitol, dextran,and mannitol, respectively.